Project 1 Summary Using the rhesus macaque (RM) model, we have repeatedly demonstrated that the SIV-specific immune responses elicited by strain 68-1 (Rh157.5/Rh157.4-deleted) RhCMV/SIV vectors (which are characterized by high frequency, broadly distributed, effector-memory CD4+ and CD8+ T cell responses) result in immediate control and ultimate clearance of mucosally-administered, highly pathogenic SIVmac239 virus in 50-60% of vaccinated RM. Although the pattern of the ?arrest and clear? protection observed in these 68-1 RhCMV/SIV vector-vaccinated RM suggests that the early pathogen intercept afforded by the vaccine-elicited ?in place? effector-memory T cells plays a critical role in efficacy, recent data indicates that this unique response characteristic is not, by itself, sufficient for protection. Indeed, we have shown that the CD8+ T cells elicited by the strain 68-1 RhCMV/SIV vectors manifest another very unusual immunologic property: all of these CD8+ T cells were found to recognize epitopes that were restricted by either MHC-II or MHC-E, not conventional MHC- Ia. We further demonstrated that this unconventional epitope targeting was reverted to conventional MHC-Ia restriction by repair of Rh157.5/Rh157.4 expression, which did not otherwise affect the functional or phenotypic characteristics of vector-elicited CD8+ T cells. Remarkably, in 2 independent studies, the Rh157.5/Rh157.4- repaired RhCMV/SIV vectors failed to protect vaccinated RM against SIV challenge, demonstrating that unconventional CD8+ T cell epitope recognition is required for RhCMV/SIV vector efficacy. Recent work has demonstrated that conventional vs. unconventional CD8+ T cell priming is regulated by multiple RhCMV genes, the modification of which effectively programs RhCMV vectors to elicit CD8+ T cell responses with distinct epitope recognition patterns. Based on these data, Projects 2-4 of this program will design and construct RhCMV and HCMV vectors that are strategically modified to both unravel the virologic mechanisms that mediate this CD8+ T cell epitope recognition ?programming?, and to develop vectors that predominantly or exclusively elicit MHC-II- vs. MHC-E-restricted CD8+ T cell responses. Project 1 will be responsible for determining the epitope recognition profile of the CD8+ T cells elicited by each of these modified vectors in RM, and for selecting RhCMV/SIV vectors that differentially elicit MHC-II- vs. MHC-E-restricted CD8+ T cell responses for efficacy testing. Project 1 will then determine the contribution of these response type(s) to efficacy and whether focusing the response on a particular response type improves efficacy, and will also define immunologic correlates of this protection. The insight gained from these studies will be used by Projects 4 and 5 to design, manufacture and clinically test a ?safety- and CD8+ T cell response-optimized? HCMV/HIV vector, and Project 1 will efficacy test the RhCMV/SIV homolog of this clinical vector in RM, using heterologous SIV challenge, to confirm efficacy and immune correlates, as well as determine the kinetics of CMV/SIV vector- mediated protection after vaccination and the dependence of efficacy on boosting and vector dose.